CA9 gene single nucleotide polymorphisms predict prognosis and treatment response of metastatic renal cell carcinoma

ABSTRACT

Methods and compositions for providing a prognosis or diagnosis for a human patient having renal cell cancer are provided. The method relates to the discovery of SNPs which are associated with a favorable prognosis and response to therapy in RCC.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/103,895 filed Oct. 8, 2008, the disclosure of whichis hereby incorporated herein by reference in its entirety for allpurposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

Not applicable

FIELD OF THE INVENTION

The present invention relates to single base polymorphisms in the CAIXgene and their use in the prognosis, diagnosis and therapy formetastatic renal cell carcinoma (MRCC).

BACKGROUND OF THE INVENTION

Every year, over 12,000 Americans succumb to metastatic renal cellcarcinoma (MRCC) [1]. The prognosis of this disease is poor and themedian survival time is only 1-2 years [2]. Chemotherapy is ineffective[3] with the notable exception of rare tumors with sarcomatoid features[4]. Until recently, cytokine-based immunotherapy was the only effectivetherapeutic approach, however, response rates were low and the treatmentwas accompanied by substantial side effects. Identification of reliablepredictors of response and survival are necessary to choose patients whomost likely benefit from these drugs. This question is now even morepertinent since the approval of sorafenib, sunitinib and temsirolimushas expanded the therapeutic options available to MRCC patients.Currently clinical and laboratory information may distinguish differentgroups [5], but molecular information such as protein [6, 7] or geneticdata [8, 9] may further improve pre-therapeutic risk assessment.

A Single Nucleotide Polymorphism (SNP) is a variation in the DNAsequence, which occurs when a nucleotide (A, T, C or G) is changed in atleast 1% of a certain population. When a SNP falls in a coding sequence,it may determine a change of an amino acid in the related proteinsequence. Such a SNP is called non-synonymous. In accordance with thedegeneracy rules of the genetic code, a SNP could also generate the sameamino acid, which is than called a synonymous SNP. Of note, severalstudies have indicated that a SNP in a non-coding region of a gene mayalso impact biological processes.

SNPs in the human genome contribute to wide variations in howindividuals respond to medications, either by changing thepharmacokinetics of drugs or by altering the cellular response totherapeutic agents [10]. Several studies have assessed the importance ofthese SNPs in predicting prognosis and response to therapies and drugs.For example, SNPs have been associated with prognosis of breast cancer,lymphoid neoplasms, and nasopharyngeal carcinoma [1]-17]. In RCC, Ito etal. [8] found that a SNP in the Signal Transducer and Activator 3 gene(STAT3) is associated with a greater likelihood of response tointerferon-alpha.

The carbonic anhydrase 9 gene (CA9) is located on chromosome 9p12-13,which represents a chromosomal area linked to prognosis in RCC [18-20].CA9 comprises 11 exons and encodes for the 459 amino acid protein CAIX.CAIX is a membrane associated protein and catalyzes the reversiblereaction H₂O+CO₂

H⁺+HCO₃ ⁻, which is crucial to a wide variety of processes including pHregulation. CAIX is not expressed in the majority of benign organs andtissues, but abundantly expressed as a direct consequence of hypoxia innumerous cancers [21]. Studies demonstrate that high CAIX expression inclear cell RCC is associated with better prognosis and a greaterlikelihood of response to IL-2 based immunotherapy [22, 23]. Takentogether, CA9 is located in a prognostically relevant chromosomal areaand is encoding for one of the most significant protein markers inmetastatic RCC. In contrast to CAIX protein, however, no efforts havebeen made to date to study the CA9 gene in metastatic RCC. Here, we testthe hypotheses that SNPs and mutations of the CA9 gene are associatedwith CAIX expression, response to immunotherapy and survival inmetastatic rCC.

At present, there are several FDA-approved drugs available for thetreatment of MRCC, namely IL-2, sunitinib, sorafenib, and temsirolimus[39-41]. Pre-therapeutic prognostic assessment is required to selectpatients most likely to benefit from certain agents. However, only a fewreliable predictors of response and survival are currently available.Motzer et al. [5] utilized clinical (performance status, time fromdiagnosis to start of therapy) and laboratory data (lactatedehydrogenase, hemoglobin, and corrected calcium levels) to predictsurvival of patients treated with interferon-alpha. Zisman et al. [42]stratified patients with MRCC into three prognostic groups based onstage, performance status and Fuhrman grade. Protein expression in thetumor and genetic information may further assist in prognosticassessment. Kim et al. [6] assessed 8 molecular markers in MRCC andfound that CAIX, PTEN, p53, and vimentin expression significantlyenhanced the predictive accuracy of a clinical prognostic model. Ito etal. [8] analyzed a cohort of 75 Japanese MRCC patients treated withinterferon-alpha. They found that rs4796743, which is located in thenon-coding 5′-flanking region of STAT3 gene, is associated with a 2.7fold greater likelihood of response to interferon-alpha.

A leading treatment for MRCC is immunotherapy with IL-2. This treatmentis associated with severe toxicities. Accordingly, there is a need toidentify patients for whom IL-2 would be of sufficient benefit towarrant the health risks. This invention provides for this need byproviding a means for identifying such patients.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to Applicant's discovery that CA9 SNPs arefrequently found in patients with MRCC. The C allele variant rs12553173in particular is associated with improved overall survival and a greaterlikelihood of response to IL-2. The Applicants have further found thatCA9 rs12553173 and CAIX expression levels are both independentprognostic factors of overall survival and complementary in predictingprognosis of MRCC.

Accordingly, in a first aspect the invention concerns a method ofproviding a prognosis for MRCC in which the presence of the C allelevariant rs12553173 is associated with a substantially improvedlikelihood of survival and response to IL-2 therapy as compared to thoseMRCC patients lacking the variant. In one preferred embodiment of theabove, the polymorphic site is the synonymous SNP rs12553173 (c.249T>C)(corresponding to conversion of a T to a C at position 187 of SEQ IDNOS:2 and 3) (SNP1).

In this aspect, the invention provides methods for providing a prognosisfor renal cell cancer (RCC) or MRCC for a human patient in need thereof,the method comprising the steps:

a) identifying the nucleotide(s) present at one or more polymorphicsites in the CA9 nucleic acid from the patient; and

b) providing the prognosis, wherein the prognosis is according to thenucleic acid identified at the polymorphic site.

In a another embodiment, the invention provides methods for providing aprognosis for renal cell cancer (RCC) or MRCC for a patient in needthereof, the method comprising the steps:

a) obtaining sample of CA9 nucleic acid from the patient;

b) identifying the nucleotide(s) present at one or more polymorphicsites in the CA9 nucleic acid; and

c) providing the prognosis, wherein the prognosis is according to thenucleic acid identified at the polymorphic site.

In other embodiments of this aspect, the CA9 nucleic acid is genomic DNAor cDNA or mRNA. In a particularly preferred embodiment, the polymorphicsite corresponds to SNP1 and the identification of the presence of a Cresidue at the SNP position indicates that the patient is a goodcandidate for, or likely to respond to, immunotherapy (e.g., therapywith IL-2). The prognosis may be provided to the patient and/or used toguide therapy. In another particularly preferred embodiment, thepolymorphic site corresponds to SNP1 and the identification of thepresence of a C residue at the SNP1 position indicates that the patienthas better odds of overall survival.

In yet another embodiment, the invention provides a method of treating apatient having RCC or MRCC, the method comprising:

a) identifying the nucleotide(s) present at one or more polymorphicsites in CA9 nucleic acid obtained from the patient; and

b) treating the patient, or selecting the patient for treatment, withimmunotherapy according to the identification of the nucleotide(s) atthe polymorphic site. In a preferred embodiment, polymorphic site isSNP1 and the identification of a C residue at the SNP1 position in theCA9 nucleic acid leads the patient to be treated, or selected fortreatment, with immunotherapy (e.g., IL-2). In preferred embodiments,the CA9 nucleic acid is genomic DNA or cDNA or mRNA.

In an other embodiment, the invention provides a method of treating apatient having RCC or MRCC, the method comprising:

a) obtaining a sample of CA9 nucleic acid from the patient;

b) identifying the nucleotide(s) present at one or more polymorphicsites in the CA9 nucleic acid; and

c) wherein the patient is treated, or selected for treatment, forimmunotherapy according to the identification of the nucleotide(s) atthe polymorphic site. In a preferred embodiment, polymorphic site isSNP1 and the identification of a C residue at the SNP1 position in theCA9 nucleic acid leads the patient to be treated, or selected fortreatment, with immunotherapy (e.g., IL-2). In preferred embodiments,the CA9 nucleic acid is genomic DNA or cDNA or mRNA.

Further embodiments of the above, the level of expression of CA9 nucleicacid or protein is also determined for a cancer tissue sample. Theexpression can be determined by measuring tissue levels of the proteinor the nucleic acid in the tissue.

The present invention also relates to allelic variants of CA9 andprovides allele-specific nucleic acid primers and probes suitable fordetecting these allelic variants for applications such as moleculardiagnosis, prediction of an individual's MRCC susceptibility andappropriate therapy, personalized medicine, and/or the genetic analysisof the CA9 gene in a population.

In a this aspect, the invention provides oligonucleotides from 10 to 40nucleotides in length each of which is identical in sequence to acorresponding sequence of CA9 which is at most 500, 400, 300, 200 or 100nucleotides from a polymorphic site. Pairs of the nucleotides are usefulin amplifying a CA9 nucleic acid comprising the polymorphic site, forexample, in a polymerase chain reaction (PCR) or in areverse-transcriptase (RT)-PCR reaction. In particular, the presentinvention provides an isolated nucleic acid comprising a sequenceidentical to that of variant CA9 gene which can be used use inidentifying the nucleotides present a the polymorphic sites. In oneembodiment of the above, the polymorphic site is the synonymous SNPrs12553173 (c.249T>C) (corresponding to conversion of a T to a C atposition 187 of SEQ ID NOS:2 and 3) (SNP1).

The invention further provides diagnostic kits comprising one or moreallele-specific oligonucleotide for SNP1, and also may include one ormore primers for use in amplifying a nucleic acid having a SNP siteaccording to the invention (e.g., SNP1).

In any of the above aspects, a preferred SNP is SNP1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Electropherograms showing wild type, heterozygous and homozygoussequences of the detected CA9 SNPs.

FIG. 2. Overall survival according to CAIX expression. The numbers ofpatients at risk are indicated.

FIG. 3. Overall survival according to CA9 SNP rs12553173 (c.249T>C). Thenumbers of patients at risk are indicated.

FIG. 4. CAIX amino acid sequence (SEQ ID NO:1).

FIG. 5. CA9 cDNA sequence (SEQ ID NO:2).

FIG. 6A-C. CA9 genomic sequence (SEQ ID NO:3).

DETAILED DESCRIPTION OF THE INVENTION

In its first aspect, the methods of the invention provide a prognosisfor a human patient having renal cell cancer (RCC) by determiningwhether the patient has the SNP1 polymorphism of a nucleic acid which isidentical or substantially identical to CA9 and providing the prognosis,wherein the presence of the SNP indicates a favorable prognosis and theabsence of the SNP indicates a less favorable prognosis. The cancer insome further embodiments is metastatic renal cell cancer (MRCC). Thedetermining can comprise the steps of obtaining a biological samplecomprising CAIX nucleic acid from the patient and then detecting thepresence of absence of the polymorphism in the nucleic acid. The nucleicacid is preferably isolated. The nucleic acid can be DNA, cDNA, mRNA, orgenomic DNA. In some embodiments, the nucleic acid is amplified by PCRor other means and a T or C residue at the SNP position is detected ordetermined in the amplified nucleic acid. In some embodiments of any ofthe above, the level of expression of a CAIX protein which is identicalor substantially identical to the CAIX protein of FIG. 4 (SEQ ID NO:1)is determined in a sample of cancerous tissue from the patient and if ahigh level of expression is found that result contributes to a favorableprognosis. Conversely a low level expression of the CAIX proteincontributes to a less favorable prognosis. The prognosis can be withrespect to the likelihood of surviving the cancer or the length ofsurvival with the cancer or both. Methods of quantitating CAIX nucleicacid or protein expression and using them in providing a prognosisand/or diagnosis are taught in U.S. patent application Ser. No.10/511,465 (assigned to the same assignee as the present application)and published as U.S. Patent Publication No. 20050158809, the contentsof which are incorporated by reference in their entirety for allpurposes and particularly with reference to such methods.

In other embodiments, the invention provides a method of treating ahuman RCC or MRCC patient by obtaining a biological sample containingCAIX nucleic acid from the patient which is identical or substantiallyidentical to a sequence of FIG. 5 (SEQ ID NO:2) or FIG. 6 (SEQ ID NO:3)and determining as above whether the nucleic acid has the SNP1polymorphism. Patients with the polymorphism are selected for treatmentwith an immunotherapy (e.g., therapy with IL-2 and/or interferon alpha).If the polymorphism is absent, the patient is treated with analternative therapy (e.g., administration of sunitinib, sorafenib, andtemsirolimus). See, de Martino et al., J Urol. 2009 August;182(2):728-34. Epub 2009 Jun. 18) which is incorporated herein byreference in its entirety.

In a second aspect, the invention provides a composition comprising afirst PCR primer which binds to DNA 3′ of a site corresponding to theSNP1 polymorphism and a second PCR primer which binds to DNA 5′ of thesite, wherein the first and second primer are complementary to nucleicacid sequences which flank the site wherein the flanking nucleic acidsequences are each within 500, 200, 100 or 50 nucleotides of the site.In a preferred embodiment, the primers are each from 10 to 22nucleotides in length. In another embodiment, the invention provides, anucleic acid probe which is complementary to a CAIX nucleic acidsequence having the SNP1 polymorphism. The probe also is preferably from12 to 22 nucleotides in length.

The invention also provides kits comprising an antibody which binds CAIXprotein and a first PCR primer which binds to DNA 3′ of a sitecorresponding to the SNP1 polymorphism and a second PCR primer whichbinds to DNA 5′ of the site, wherein the first and second primer arecomplementary to nucleic acid sequences which flank the site wherein theflanking nucleic acid sequences are each within 500 nucleotides of thesite; or a nucleic acid probe which is complementary to a CAIX nucleicacid sequence having the SNP1 polymorphism.

In some embodiments, the invention relates to a method of aiding in arenal cell carcinoma prognosis that includes in addition to identifyingthe presence or absence of a SNP in the patient also (a) quantifyingexpressed carbonic anhydrase IX (CAIX), if any, present in one or moresamples derived from a subject diagnosed with renal cell carcinoma(e.g., renal clear cell carcinoma) to produce quantified CAIX expressiondata. The method also includes (b) correlating the quantified CAIXexpression data with a probability of a renal cell carcinoma prognosisfor the subject. The expressed CAIX typically includes a CAIXpolypeptide, a fragment of a CAIX polypeptide, an mRNA that encodes aCAIX polypeptide, or the like. Although other quantification techniquesare optionally utilized, in preferred embodiments, the expressed CAIX isquantified by immunohistochemical staining. In addition, the samples aregenerally derived from a renal tumor and/or a metastatic lesion derivedfrom a renal tumor.

Quantified CAIX expression data correlates with various outcomes for RCCpatients. For example, when the quantified CAIX expression datacomprises a quantification percentage of more than 85% thatquantification percentage correlates with a better prognosis for thesubject than a quantification percentage of 85% or less when the subjectis diagnosed with metastatic renal cell carcinoma. Further, when thequantified CAIX expression data comprises a quantification percentage of85% or less that quantification percentage correlates with a betterprognosis for the subject than a quantification percentage of 85% orless when the subject is diagnosed with non-metastatic renal cellcarcinoma of T stage≧3 and Fuhrman grade≧2.

The method additionally identifies RCC patients that may benefit fromparticular courses of treatment. To illustrate, when the quantified CAIXexpression data comprises a quantification percentage of more than 85%that quantification percentage further correlates with a likely positiveresponse to, e.g., interleukin-2 (IL-2) immunotherapy, or one or moreCAIX-targeted therapies, for the subject. In addition, when thequantified CAIX expression data comprises a quantification percentage of85% or less that quantification percentage further correlates with alikely positive response to an adjuvant immunotherapy for the subjectwhen the subject is diagnosed with non-metastatic renal cell carcinomaof T stage≧3 and Fuhrman grade≧2.

In another aspect, the invention relates to a method of aiding in arenal clear cell carcinoma prognosis that includes (a) quantifyingexpressed CAIX polypeptides, if any, present in one or more samplesderived from a subject diagnosed with renal clear cell carcinoma toproduce quantified CAIX polypeptide expression data in which the samplesare derived from a renal tumor and/or a metastatic lesion derived from arenal tumor. The method also includes (b) correlating the quantifiedCAIX polypeptide expression data with a probability of a renal clearcell carcinoma prognosis in which a quantification percentage of 85%stratifies the prognosis for the subject. In preferred embodiments, theexpressed CAIX polypeptides are quantified by immunohistochemicalstaining and the quantification percentage comprises a positive stainingpercentage.

The quantified CAIX expression data produced with this method alsocorrelates with various outcomes for RCC patients and further identifiesRCC patients that may need specific courses of treatment. For example, aquantification percentage of more than 85% correlates with a betterprognosis for the subject than a quantification percentage of 85% orless when the subject is diagnosed with metastatic renal clear cellcarcinoma, or when the subject is diagnosed with non-metastatic renalclear cell carcinoma of T stage.gtoreq.3 and Fuhrman grade.gtoreq.2. Aquantification percentage of more than 85% for a sample derived from therenal tumor correlates with a lower probability of metastasis than aquantification percentage of 85% or less for the sample derived from therenal tumor. In addition, a quantification percentage of more than 85%further correlates with a likely positive response to interleukin-2immunotherapy for the subject, or with a likely positive response to oneor more CAIX-targeted therapies for the subject. Moreover, aquantification percentage of 85% or less further correlates with alikely positive response to an adjuvant immunotherapy for the subjectwhen the subject is diagnosed with non-metastatic renal cell carcinomaof T stage≧3 and Fuhrman grade≧2.

In certain embodiments of the methods described herein, the quantifiedCAIX expression data are in a computer-readable form. In theseembodiments, (b) typically comprises operating a programmable computerthat comprises at least one database and executing an algorithm thatdetermines closeness-of-fit between the computer-readable quantifiedCAIX expression data and database entries, which entries correspond toclinical and/or pathological data for a population of renal carcinomapatients (e.g., renal clear cell carcinoma patients) to therebycorrelate the quantified CAIX expression data with the probability ofthe renal carcinoma prognosis (e.g., renal clear cell carcinomaprognosis) for the subject.

In yet another aspect, the present invention provides a computer programproduct comprising a computer readable medium having one or more logicinstructions. The computer readable medium includes logic instructionsfor (a) receiving quantified CAIX expression data derived from a subjectdiagnosed with renal cell carcinoma. The computer readable medium alsoincludes logic instructions for (b) determining closeness-of-fit betweenthe quantified CAIX expression data and database entries, which entriescorrespond to clinical and/or pathological data for a population ofrenal cell carcinoma patients to thereby correlate the quantified CAIXexpression data with a probability of a renal cell carcinoma prognosisfor the subject.

The CAIX antigen is typically quantitated in mammalian samples, whichare preferably human samples. Such samples optionally include tissuespecimens, body fluids (e.g., urine), tissue extracts, cells, celllysates and cell extracts, among other samples. In preferredembodiments, samples are derived from renal tumors and/or metastaticlesions derived from renal tumors.

The CAIX antigen can be detected and quantified by various techniques.In preferred embodiments, CAIX is detected and quantified byimmunohistochemical staining (e.g., using tissue arrays or the like).Preferred tissue specimens to assay by immunohistochemical staining, forexample, include cell smears, histological sections from biopsiedtissues or organs, and imprint preparations among other tissue samples.An exemplary immunohistochemical staining protocol is described furtherbelow. Such tissue specimens can be variously maintained, for example,they can be fresh, frozen, or formalin-, alcohol- or acetone- orotherwise fixed and/or paraffin-embedded and deparaffinized. Biopsiedtissue samples can be, for example, those samples removed by aspiration,bite, brush, cone, chorionic villus, endoscopic, excisional, incisional,needle, percutaneous punch, and surface biopsies, among other biopsytechniques.

As mentioned, many formats for detection and quantification of the CAIXantigen are optionally adapted for use with the methods of the presentinvention. Certain exemplary techniques include, e.g., Western blotting,immunoassays (e.g., radioimmunoassays (RIAs), enzyme immunoassays(EIAs), etc.), immunohistochemical staining, immunoelectron and scanningmicroscopy using immunogold, ELISAs, competitive EIA or dual antibodysandwich assays, among other assays commonly known in the art.

Representative of one type of ELISA test for CAIX antigen is a format inwhich a microtiter plate is coated with antibodies made to CAIXpolypeptides or antibodies made to whole cells expressing CAIX proteins,and to this is added a patient sample, for example, a tissue or cellextract. After a period of incubation permitting any antigen to bind tothe antibodies, the plate is washed and another set of anti-CAIXantibodies which are linked to an enzyme is added, incubated to allowreaction to take place, and the plate is then rewashed. Thereafter,enzyme substrate is added to the microtiter plate and incubated for aperiod of time to allow the enzyme to work on the substrate, and theabsorbance of the final preparation is measured. A large change inabsorbance typically indicates a positive result.

It is also apparent to one skilled in the art of immunoassays that CAIXpolypeptides can be used to detect and/or quantitate the presence ofCAIX antigen in the body fluids, tissues and/or cells of patients. Inone such embodiment, a competition immunoassay is used, wherein the CAIXprotein is labeled and a body fluid is added to compete with the bindingof the labeled CAIX polypeptide to antibodies specific to CAIXpolypeptide.

As another exemplary embodiment, an immunometric assay may be used inwhich a labeled antibody made to a CAIX protein is used. In such anassay, the amount of labeled antibody which complexes with theantigen-bound antibody is directly proportional to the amount of CAIXantigen in the sample.

Antibodies suitable for use in certain embodiments of the methodsdescribed herein may be prepared by conventional methodology and/or bygenetic engineering. Antibody fragments may be genetically engineered,preferably from the variable regions of the light and/or heavy chains(V_(H) and V_(L)), including the hypervariable regions, and still morepreferably from both the V_(H) and V_(L) regions. For example, the term“antibodies” as used herein includes polyclonal and monoclonalantibodies and biologically active fragments thereof including amongother possibilities “univalent” antibodies (Glennie et al. (1982) Nature295:712); Fab proteins including Fab′ and F(ab′)₂ fragments whethercovalently or non-covalently aggregated; light or heavy chains alone,preferably variable heavy and light chain regions (V_(H) and V_(L)regions), and more preferably including the hypervariable regions(otherwise known as the complementarity determining regions (CDRs) ofthe V_(H) and V_(L) regions); F_(c) proteins; “hybrid” antibodiescapable of binding more than one antigen; constant-variable regionchimeras; “composite” immunoglobulins with heavy and light chains ofdifferent origins; “altered” antibodies with improved specificity andother characteristics as prepared by standard recombinant techniques andalso by oligonucleotide-directed mutagenesis techniques(Dalbadie-McFarland et al. (1982) Proc. Natl. Acad. Sci. USA 79: 6409).

The antibodies useful according to this invention to identify CAIXpolypeptides can be labeled in essentially any manner, for example, withenzymes such as horseradish peroxidase (HRP), fluorescent compounds, orwith radioactive isotopes such as, ¹²⁵I, among other labels.

Bispecific antibodies that are optionally adapted for use in the presentinvention can be produced by chemically coupling two antibodies of thedesired specificity. Bispecific MAbs can preferably be developed bysomatic hybridization of 2 hybridomas. Bispecific MAbs for targetingCAIX protein and another antigen can be produced by fusing a hybridomathat produces CAIX-specific MAbs with a hybridoma producing MAbsspecific to another antigen. For example, a cell (a quadroma), formed byfusion of a hybridoma producing a CAIX-specific MAb and a hybridomaproducing an anti-cytotoxic cell antibody, will produce hybrid antibodyhaving specificity of the parent antibodies. See, e.g., Immunol. Rev.(1979); Cold Spring Harbor Symposium Quant. Biol., 41: 793 (1977); vanDijk et al., Int. J. Cancer, 43: 344-349 (1989). Thus, a hybridomaproducing a CAIX-specific MAb can be fused with a hybridoma producing,for example, an anti-T3 antibody to yield a cell line which produces aCAIX/T3 bispecific antibody which can target cytotoxic T cells toCAIX-expressing tumor cells.

Although representative hybridomas of use in practicing this inventionare formed by the fusion of murine cell lines, human/human hybridomas(Olsson et al. (1980) Proc. Natl. Acad. Sci. USA 77:5429) andhuman/murine hybridomas (Schlom et al. (1980) Proc. Natl. Acad. Sci. USA77:6841; Shearman et al. (1991) J. Immunol. 146: 928-935; and Gorman etal. (1991) Proc. Natl. Acad. Sci. USA 88:4181-4185) can also be preparedamong others.

Monoclonal antibodies for use in the methods of this invention may beobtained by methods well known in the art. See, e.g., Galfre andMilstein, “Preparation of Monoclonal Antibodies: Strategies andProcedures,” in Methods in Enzymology: Immunochemical Techniques, 73:1-46 [Langone and Vanatis (eds); Academic Press (1981)]. See also,Milstein and Kohler (1975) Nature 256:495-497. Monoclonal antibodiesspecific for this invention can be prepared by immunizing appropriatemammals, preferably rodents, rabbits or mice, with an appropriateimmunogen, for example, MaTu-infected HeLa cells, CAIX fusion proteins,or CAIX proteins attached to a carrier protein, if necessary.

Representative MAbs of use in this invention include MAbs M75, MN9, MN12and MN7. For example, Monoclonal antibody M75 (MAb M75) is produced bymouse lymphocytic hybridoma VU-M75, which was initially deposited in theCollection of Hybridomas at the Institute of Virology, Slovak Academy ofSciences (Bratislava, Slovakia) and was deposited under ATCC DesignationHB 11128 on Sep. 17, 1992 at the American Type Culture Collection(ATCC). The production of hybridoma VU-M75 is described in Zavada etal., International Publication No. WO 93/18152. Mab M75 recognizes boththe nonglycosylated GST-MN fusion protein and native CAIX protein asexpressed in CGL3 cells equally well. The M75 MAb recognizes both nativeand denatured forms of the CAIX protein (Pastorekova et al. (1992)Virology 187:620-626).

Antibodies employed in assays may be labeled or unlabeled. Unlabeledantibodies may be employed in agglutination; labeled antibodies may beemployed in a wide variety of assays, employing a wide variety of labelsknown in the art. Suitable detection means include the use of labelssuch as radionuclides, enzymes, coenzymes, fluorescers,chemiluminescers, chromogens, enzyme substrates or co-factors, enzymeinhibitors, free radicals, particles, dyes and the like. Such labeledreagents may be used in a variety of well known assays (referred toabove), such as radioimmunoassays, enzyme immunoassays, e.g., ELISA,fluorescent immunoassays, and the like. See, e.g., U.S. Pat. Nos.3,766,162; 3,791,932; 3,817,837; and 4,233,402.

An exemplary immunohistochemical staining protocol using a Dako stainingkit (Dako Corporation, Carpenteria, Calif.) includes dewaxing,rehydrating and blocking sample sections to remove non-specificreactivity as well as endogenous peroxidase activity. Sections can thenbe incubated with dilutions of the M75 monoclonal antibody. After theunbound M75 is removed by rinsing the section, the section can besequentially reacted with a biotinylated antimouse IgG antibody andstreptavidin conjugated to horseradish peroxidase; a rinsing step can beincluded between those two reactions and after the second reaction.Following the last rinse, the antibody-enzyme complexes can be detectedby reaction with an insoluble chromogen (diaminobenzidine) and hydrogenperoxide. A positive result is indicated by the formation of aninsoluble reddish-brown precipitate at the site of the primary antibodyreaction. The sections can then be rinsed, counterstained withhematoxylin, dehydrated and cover slipped. Thereafter, the sections canbe examined using standard light microscopy. A deposit of a reddishbrown precipitate over the plasma membrane is evidence that the M75antibody has bound to a CAIX antigen in the tissue. A known positivecontrol (e.g., CGL3) can be stained to validate the assay. Sectionthickness should be taken into consideration when comparing stainingintensities, as thicker sections produce greater staining intensityindependent of other assay parameters.

In certain embodiments of the invention, mRNA that encodes a CAIXpolypeptide is optionally detected in a sample and correlated with aprognosis for a patient. Detection of RNA transcripts may be achieved byNorthern blotting, for example, in which a preparation of RNA is run ona denaturing agarose gel, and transferred to a suitable support, such asactivated cellulose, nitrocellulose or glass or nylon membranes.Radiolabelled cDNA or RNA is then hybridized to the preparation, washedand analyzed by autoradiography. In situ hybridization visualization mayalso be employed in which a radioactively labelled antisense cRNA probeis hybridized with a thin section of a biopsy sample, washed, cleavedwith RNase and exposed to a sensitive emulsion for autoradiography. Thesamples may be stained with haematoxylon to demonstrate the histologicalcomposition of the sample, and dark field imaging with a suitable lightfilter illuminates the developed emulsion. Non-radioactive labels suchas digoxigenin may also be used.

General texts describing additional molecular biological techniquesuseful herein, including the preparation of antibodies include Bergerand Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology, Vol. 152, Academic Press, Inc., Sambrook et al., MolecularCloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring HarborLaboratory (1989), Current Protocols in Molecular Biology, F. M. Ausubelet al. (Eds.), Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc. (supplementedthrough 2000), Harlow et al., Monoclonal Antibodies: A LaboratoryManual, Cold Springs Harbor Laboratory Press (1988), Paul (Ed.),Fundamental Immunology, Lippincott Williams & Wilkins (1998), and Harlowet al., Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press (1998).

Following detection and quantitation of CAIX in one or more samples froma subject diagnosed with RCC, the SNP data and CAIX expression data iscorrelated with clinical and/or pathological data to arrive atprognostic information for the patient. Data generated by the methodsdescribed herein is optionally analyzed using any suitable technique.Statistical analysis of data and more particularized correlations aredescribed in greater detail in an example provided below. In oneembodiment, data is analyzed with the use of a logic device, such as aprogrammable digital computer that is included, e.g., as part of asystem. The computer generally includes a computer readable medium thatstores logic instructions of the system software. Certain logicinstructions are typically devoted to memory for receiving quantifiedCAIX expression data derived from a subject diagnosed with renal cellcarcinoma. The computer also typically includes logic instructions fordetermining closeness-of-fit between the quantified CAIX expression dataand database entries, which entries correspond to clinical and/orpathological data for a population of renal cell carcinoma patients tothereby correlate the quantified CAIX expression data with a probabilityof a renal cell carcinoma prognosis for the subject.

In preferred embodiments, the quantified CAIX expression data is in acomputer-readable form suitable for use in database queries. Forexample, a database query generally includes operating a programmablecomputer that comprises at least one database and executing an algorithmthat determines closeness-of-fit between the computer-readablequantified CAIX expression data and database entries, which entriescorrespond to clinical and/or pathological data for a population ofrenal clear cell carcinoma patients to thereby correlate the quantifiedCAIX expression data with the probability of the renal clear cellcarcinoma prognosis for the subject. In some embodiments, the algorithmincludes an artificial intelligence algorithm or a heuristic learningalgorithm. For example, the artificial intelligence algorithm optionallyincludes one or more of, e.g., a fuzzy logic instruction set, a clusteranalysis instruction set, a neural network, a genetic algorithm, or thelike.

The present invention also provides a computer program productcomprising a computer readable medium having one or more logicinstructions. The computer readable medium includes logic instructionsfor receiving the SNP data and (a) receiving quantified CAIX expressiondata derived from a subject diagnosed with renal cell carcinoma. Thecomputer readable medium also includes logic instructions for (b)determining closeness-of-fit between the quantified CAIX expression dataand database entries, which entries correspond to clinical and/orpathological data for a population of renal cell carcinoma patients tothereby correlate the quantified CAIX expression data with a probabilityof a renal cell carcinoma prognosis for the subject. Furthermore, thecomputer readable medium optionally includes, e.g., a CD-ROM, a floppydisk, a tape, a flash memory device or component, a system memory deviceor component, a hard drive, or a data signal embodied in a carrier wave.

In some embodiments, the presence or absence of VHL gene mutation in thecancer tissue sample is further determined to aid in the prognosis. Theabsence of the VHL mutation and low CAIX expression are associated withtumor aggressiveness and poor survival of clear cell renal cellcarcinoma (see, Patard, et al., Int J Cancer. 2008 July; 123(2):395-400. Both CAIX expression and VHL mutational status are able tostratify patients with clear cell RCC into distinct groups with regardsto clinicopathological variables and prognosis, with low CAIX expressionand absence of VHL mutation being associated with a poorclinicopathological phenotype and diminished survival. Combination ofCAIX expression and VHL mutational status further enhances prognosticstratification: patients with both VHL mutation and high CAIX expressionhave the most favorable prognosis, patients with either VHL mutation orhigh CAIX expression have intermediate prognosis, and patients withneither VHL mutation nor high CAIX expression have the worst prognosis.The findings can be used for patient selection for targeted therapy. VHLalteration and inactivation through mutation or hypermethylation occursin more than 50% of sporadic clear cell RCCs. VHL alteration is directlylinked to tumorigenesis via the hypoxia-induced pathway, which leads toover-expression of several important proteins such as VEGF and CAIX.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

All publications, patents, patent applications, databases and otherreferences cited in this application are herein incorporated byreference in their entirety as if each individual publication, patent,patent application, database or other reference was specifically andindividually indicated to be incorporated by reference to the extentthat each is not inconsistent with the present disclosure.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural reference unless the contextclearly dictates otherwise.

“Renal cell carcinoma” or “RCC” refers to carcinoma of the renalparenchyma. RCC is also often identified as renal cancer,“hypemephroma”, or adenocarcinoma of the kidney. There are four maintypes of renal cell carcinoma, namely, clear cell type, granular celltype, mixed granular and clear cell type, and spindle cell type.

“Prognosis” refers to a forecast as to the probable outcome of a diseasestate, a determination of the prospect as to recovery from a disease asindicated by the nature and symptoms of a case, the monitoring of thedisease status of a patient, the monitoring of a patient for recurrenceof disease, and/or the determination of the preferred therapeuticregimen for a patient.

“Quantification percentage” refers to a CAIX expression score thatincludes the percentage of a sample (e.g., a target tissue or cellularsample, such as a sample from a renal tumor, a sample from a metastaticlesion derived from a metastitic lesion, and/or the like) that haspositive CAIX expression. In preferred embodiments, the quantificationpercentage of a sample refers a CAIX expression score that includes theextent of staining or staining percentage (e.g., the percentage of cellsin a sample that stain positively for CAIX, etc.). In certainembodiments, other factors such as staining intensity and the percentagestaining at maximal staining intensity are also included in a CAIXexpression score for a particular sample. For example, as illustrated inan example provided below, survival tree analysis of CAIX scoringinformation from the analyzed tissue arrays identified that a stainingpercentage of 85% was an ideal cutoff for stratification for patientsurvival. Staining percentages>85%, irrespective of intensity, wereconsidered high CAIX staining, whereas those .ltoreq.85% were consideredlow CAIX staining

As used herein nucleic acid, polynucleotide and oligonucleotide are usedinterchangeably and refer to a polymeric (e.g., 2 or more monomers) ofnucleotides of any length. A nucleic acid can be DNA, RNA, mRNA, orcDNA, and be single- or double-stranded. Oligonucleotides can benaturally occurring nucleotides or synthetic nucleotides, but aretypically prepared by synthetic means. Preferred nucleic acids of theinvention include segments of DNA or their complements including anucleotide having a sequence identical or completely complementary tothe sequence of SEQ ID NO: 2 or 3 about the SNP1 site or includingsequences identical or completely complementary to a sequence of SEQ IDNO:2 or 3 in which the sequences includes the position of a SNP or a SNPset forth therein (e.g., SNP1). The segments are usually between 10 and100 contiguous bases, and often range from about 12 to 30, 15 to 30, or20 to 30 nucleotides or from about 20 to about 50 nucleotides. Thenucleic acid bases are typically selected from G, C, T, U, and A. Somenucleic acids contain one or a plurality of polymorphic sites and haveone, or two or more polymorphic sites.

Generally, an isolated SNP-containing nucleic acid molecule comprisesone or more SNP positions disclosed by the present invention withflanking nucleotide sequences on either side of the SNP positions. Aflanking sequence can include nucleotide residues that are naturallyassociated with the SNP site and/or heterologous nucleotide sequences.Preferably the flanking sequence is up to about 500, 300, 100, 60, 50,30, 25, 20, 15, 10, 8, or 4 nucleotides (or any other length in-between)on either side of a SNP position, or as long as the full-length gene orentire protein-coding sequence (or any portion thereof such as an exon).

For full-length genes and entire protein-coding sequences, a SNPflanking sequence can be, for example, up to about 3 KB, 2 KB, 1 KB oneither side of the SNP. Furthermore, in such instances, the isolatednucleic acid molecule comprises exonic sequences (includingprotein-coding and/or non-coding exonic sequences), but may also includeintronic sequences. Thus, any protein coding sequence may be eithercontiguous or separated by introns. The important point is that thenucleic acid is isolated from flanking sequences of appropriate lengthsuch that it can be subjected to the specific manipulations or usesdescribed herein such as preparation of probes and primers for assayingthe SNP position, and other uses specific to the SNP-containing nucleicacid sequences.

An isolated SNP-containing nucleic acid molecule can comprise, forexample, a full-length gene or transcript, such as a gene isolated fromgenomic DNA (e.g., by cloning or PCR amplification), a cDNA molecule, oran mRNA transcript molecule. Polymorphs are set forth in Table 3.Furthermore, fragments of such full-length genes and transcripts thatcontain one or more SNPs disclosed herein are also encompassed by thepresent invention, and such fragments may be used, for example, toexpress any part of a protein, such as a particular functional domain oran antigenic epitope.

An isolated SNP-containing nucleic acid molecule can comprise, forexample, a full-length gene or transcript, such as a gene isolated fromgenomic DNA (e.g., by cloning or PCR amplification), a cDNA molecule, oran mRNA transcript molecule. Polymorphs are set forth in Table 3.Furthermore, fragments of such full-length genes and transcripts thatcontain one or more SNPs disclosed herein are also encompassed by thepresent invention, and such fragments may be used, for example, toexpress any part of a protein, such as a particular functional domain oran antigenic epitope.

Thus, the present invention also encompasses fragments of the nucleicacid sequences. A fragment typically comprises a contiguous nucleotidesequence at least about 8 or more nucleotides, more preferably at leastabout 12 or more nucleotides, and even more preferably at least about 16or more nucleotides. Further, a fragment could comprise at least about18, 20, 22, 25, 30, 40, 50, 60, 80, 100, 150, 200, 250 or 500 (or anyother number in-between) nucleotides in length. The length of thefragment will be based on its intended use as a polynucleotide probe orprimer. A labeled probe can then be used, for example, to screen a cDNAlibrary, genomic DNA library, or mRNA to isolate nucleic acidcorresponding to the coding region. Further, primers can be used inamplification reactions, such as for purposes of assaying one or moreSNPs sites or for cloning specific regions of a CAIX gene.

An isolated nucleic acid molecule of the present invention furtherencompasses a SNP-containing polynucleotide that is the product of anyone of a variety of nucleic acid amplification methods, which are usedto increase the copy numbers of a polynucleotide of interest in anucleic acid sample. Such amplification methods are well known in theart, and they include but are not limited to, polymerase chain reaction(PCR) (U.S. Pat. Nos. 4,683,195; and 4,683,202; PCR Technology:Principles and Applications for DNA Amplification, ed. H. A. Erlich,Freeman Press, NY, N.Y., 1992), ligase chain reaction (LCR) (Wu andWallace, Genomics 4:560, 1989; Landegren et al., Science 241:1077,1988), strand displacement amplification (SDA) (U.S. Pat. Nos.5,270,184; and 5,422,252), transcription-mediated amplification (TMA)(U.S. Pat. No. 5,399,491), linked linear amplification (LLA) (U.S. Pat.No. 6,027,923), and the like, and isothermal amplification methods suchas nucleic acid sequence based amplification (NASBA), and self-sustainedsequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874, 1990). Based on such methodologies, a person skilled in the artcan readily design primers in any suitable regions 5′ and 3′ to a SNPdisclosed herein. Such primers may be used to amplify DNA of any lengthso long that it contains the SNP of interest in its sequence.

As used herein, an “amplified polynucleotide” of the invention is aSNP-containing nucleic acid molecule whose amount has been increased atleast five-fold by any nucleic acid amplification method performed invitro as compared to its starting amount in a test sample. In otherpreferred embodiments, an amplified polynucleotide is the result of atleast ten fold, fifty fold, one hundred fold, one thousand fold, or eventen thousand fold increase as compared to its starting amount in a testsample. In a typical PCR amplification, a polynucleotide of interest isoften amplified at least fifty thousand fold in amount over theunamplified genomic DNA, but the precise amount of amplification neededfor an assay depends on the sensitivity of the subsequent detectionmethod used.

Generally, an amplified polynucleotide is at least about 16 nucleotidesin length. More typically, an amplified polynucleotide is at least about20 nucleotides in length. In a preferred embodiment of the invention, anamplified polynucleotide is at least about 30 nucleotides in length. Ina more preferred embodiment of the invention, an amplifiedpolynucleotide is at least about 32, 40, 45, 50, or 60 nucleotides inlength. In yet another preferred embodiment of the invention, anamplified polynucleotide is at least about 100, 200, 300, 400, or 500nucleotides in length. While the total length of an amplifiedpolynucleotide of the invention can be as long as an exon, an intron orthe entire gene where the SNP of interest resides, an amplified productcan be up to about 1,000 nucleotides in length (although certainamplification methods may generate amplified products greater than 1000nucleotides in length). More preferably, an amplified polynucleotide isnot greater than about 600-700 nucleotides in length. It is understoodthat irrespective of the length of an amplified polynucleotide, a SNP ofinterest may be located anywhere along its sequence.

In a specific embodiment of the invention, the amplified productcontains a SNP disclosed herein (e.g., SNP1).

The present invention provides isolated nucleic acid molecules thatcomprise, consist of, or consist essentially of one or morepolynucleotide sequences that contain one or more SNPs disclosed herein,complements thereof, and SNP-containing fragments thereof.

Although nucleotides are usually joined by phosphodiester linkages, theterm also includes polymeric nucleotides containing neutral amidebackbone linkages composed of aminoethyl glycine units. This term refersonly to the primary structure of the molecule. Thus, this term includesdouble- and single-stranded DNA and RNA. It also includes known types ofmodifications, for example, labels, methylation, “caps”, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.), those containing pendant moieties, including, forexample, proteins (including for e.g., nucleases, toxins, antibodies,signal peptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide.Polynucleotides include both sense and antisense strands.

Sequence means the linear order in which monomers occur in a polymer,for example, the order of amino acids in a polypeptide or the order ofnucleotides in a polynucleotide.

A complementary nucleotide sequence is one which allows binding to thereference nucleotide sequence in a sequence specific manner understringent conditions. A complementary sequence is usually at least 90%,95%, 96%, 97%, 98%, or 99% identical (or completely identical) to areferenced sequence. Complementary sequences include completelycomplementary sequences as determined by application of the Watson-Crickbase pairing rules such that the bases G, A, and T of the first nucleicacid are respectively and consistently paired with the bases C, U, and Aof the second or reference nucleic acid (e.g., 5′-A-G-T-C-3′ base pairswith 3′-T-C-A-G-S′).

As used herein, the term isolated, refers to a nucleic acid orpolypeptide which is separated from other nucleic acid molecules,polypeptides, or cellular materials when such were present in the sourceof the nucleic acid molecule or polypeptide. An “isolated” nucleic acidmolecule, for example, a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. An isolated entity is removedtypically from at least a majority of the differing constituents of thesource as evaluated by total mass of the differing constituents in amedium. In other words, it is typically has at most one-half, one-fourthor one-eighth the total contaminants as the source material. In apreferred embodiment, a nucleic acid molecule encoding a singlenucleotide polymorphism of the invention or including the position of asingle nucleotide polymorphism of the invention is isolated. In anotherpreferred embodiment, the SNP of the isolated nucleotide is SNP1.

“SNP” refers to a single nucleotide polymorphism in a gene sequence. TheSNP can occur in any region of the gene, including the promoter region,untranslated 5′ and 3′ regions, introns, and coding regions found in themRNA. Single nucleotide polymorphism (SNP) analysis is useful fordetecting differences between alleles of the CAIX polynucleotides (e.g.,genes) of the invention.

“CAIX or CA9” with reference to nucleic acids, e.g., gene, pre-mRNA,mRNA, and polymorphic variants, alleles, mutants concerns human nucleicacid sequences having greater than about 95%, preferably greater thanabout 96%, 97%, 98%, 99%, or higher nucleotide sequence identity,preferably over a region of at least about 25, 50, 100, 150, 200, 250,500, 1000, or more nucleotides, to the referenced nucleic acid sequence(e.g., SEQ ID NOS:2 and 3). The sequence may differ only be a referencedSNP or a combination of the SNPs disclosed herein.

“Nucleic acid” refers to polymers of deoxyribonucleotides andribonucleotides in either single- or double-stranded form, and thecomplements thereof. In the context of primers and probes, the termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

A particular nucleic acid sequence also implicitly encompasses “splicevariants.” Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant of thatnucleic acid. “Splice variants,” as the name suggests, are products ofalternative splicing of a gene. After transcription, an initial nucleicacid transcript may be spliced such that different (alternate) nucleicacid splice products encode different polypeptides. Mechanisms for theproduction of splice variants vary, but include alternate splicing ofexons. Alternate polypeptides derived from the same nucleic acid byread-through transcription are also encompassed by this definition. Anyproducts of a splicing reaction, including recombinant forms of thesplice products, are included in this definition. An example ofpotassium channel splice variants is discussed in Leicher, et al., J.Biol. Chem. 273(52):35095-35101 (1998).

“CAIX or CA9” with reference to polypeptides and proteins concerns humanpolypeptides having an amino acid sequence that has greater than about90% amino acid sequence identity, preferably 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% or greater amino acid sequence identity, preferablyover a region of over a region of at least about 25, 50, 100, 200, 500,or more amino acids, to a polypeptide encoded by a referenced CAIXnucleic acid or an amino acid sequence described herein, for example, asdepicted in SEQ ID NO:1. The nucleic acids and proteins of the inventioninclude both isolated and recombinant molecules.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterm “amino acid” refers to naturally occurring. Naturally occurringamino acids are those encoded by the genetic code, as well as thoseamino acids that are later modified, e.g., hydroxyproline,y-carboxyglutamate, and O-phosphoserine. Amino acids may be referred toherein by either their commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes.

Linkage disequilibrium or allelic association denotes a preferentialassociation of a particular allele or genetic marker with a specificallele, or genetic marker at a nearby chromosomal location morefrequently than expected by chance for the particular allelicfrequencies in the population. To illustrate, let locus A have allelesa₁ and a₂, which occur equally frequently. Let A be linked to locus Bhaving alleles b₁ and b₂, which occur equally frequently. The haplotypea_(1b1) ought to have a frequency of 0.25 in the population. If a_(1b1)occurs more frequently, then alleles a₁ and b₁ are in linkagedisequilibrium. Linkage disequilibrium may result from natural selectionor because an allele is too new to have achieved equilibrium with thelinked allele.

Linkage disequilibrium markers can be used to detect a trait even whenthe marker itself does not cause the trait. To illustrate, a marker (A)that is not a cause of a trait, but which is in linkage disequilibriumwith a gene (B) causing the trait, can be used to detect a trait toindicate susceptibility to the trait even when the gene A may not havebeen identified or detected. Newer alleles (i.e., arising from mutationrelatively recently) are expected to have a larger genomic sequencementin linkage disequilibrium. The age of an allele can be determined bycomparing its occurrence between ethnic human groups and/or betweenhumans and related species.

Hybridization probes capable of binding in a base-specific manner to aSNP site of a completely complementary strand of nucleic acid are alsoprovided by the invention. Such probes include nucleic acids and peptidenucleic acids, as described in Nielsen et al., Science 254, 1497-1500(1991). Hybridizations are usually performed under stringenthybridization conditions. Stringent hybridization conditions typicallyrefers to conditions under which a probe will hybridize to its targetsubsequence, typically in a complex mixture of nucleic acid, but to noother sequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, highly stringent conditions are selectedto be about 5-10° C. lower than the thermal melting point (T_(m)) forthe specific sequence at a defined ionic strength pH. Low stringencyconditions are generally selected to be about 15-30° C. below the T_(m).The T_(m) is the temperature (under defined ionic strength, pH, andnucleic concentration) at which 50% of the probes complementary to thetarget hybridize to the target sequence at equilibrium (as the targetsequences are present in excess, at T_(m), 50% of the probes areoccupied at equilibrium). Stringent conditions will be those in whichthe salt concentration is less than about 1.0 M sodium ion, typicallyabout 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes(e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g., greater than 50 nucleotides). Stringent conditions may also beachieved with the addition of destabilizing agents (e.g., formamide).For selective or specific hybridization, a positive signal is at leasttwo times background, preferably 10 times background hybridization.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

“Biological sample” is used in its broadest sense to reference samplesfrom a patient containing CAIX nucleic acid or protein. They sample maycomprise a bodily fluid including, but not limited to, ascites, blood,serum, plasma, platelets, saliva, cerebrospinal fluid, lymph, semen,sputum, urine and the like; the soluble fraction of a cell preparation,or an aliquot of media in which cells were grown; a chromosome, anorganelle, or membrane isolated or extracted from a cell; genomic DNA,mRNA, or cDNA in solution or bound to a substrate; a cell; a tissuepatient tissue, (e.g., sections of tissues such as cancer biopsysamples, frozen sections taken for histologic purposes), a tissuebiopsy, or a tissue print; buccal cells, skin, hair, a hair follicle;and the like.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like).Such sequences are then said to be “substantially identical.” Thisdefinition also refers to, or may be applied to, the compliment of atest sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions. Asdescribed below, the preferred algorithms can account for gaps and thelike. Preferably, identity exists over a region that is at least about25 amino acids or nucleotides in length, or more preferably over aregion that is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term encompasses nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al., John Wiley& Sons.

For PCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al. (1990) PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y.).

The increased likelihood of an individual having a trait (responder/nonresponder to a therapy or favorable/unfavorable outcome with respect tosurvival) is with reference to a population of individuals who do notharbor the polymorphic form associated with the likelihood of having thetrait. Generally, the increased likelihood can be assessed in terms ofan odds ratio which compares the frequency of a polymorphism in apopulation having a trait to a well-matched control population nothaving the trait. Odds ratios that are greater than 1 are generallyindicative of a trait being associated with a polymorphism. The greaterthe odds ratio, the greater the risk. In some embodiments, the SNP isassociated with odds ratios of at least 1.4, 1.5, 1.8, 2, 3, or 5 for aninflammatory disorder, immune system disorder or a cell proliferationdisorder. Such SNPs can be particularly useful in assessing the risk ofdeveloping such a disorder or the increased likelihood of a personhaving such a SNP responding therapeutic or prophylactic treatment.

To amplify a target SNP, the nucleic acid encoding the SNP is madeaccessible to the components of the amplification system. In general,this accessibility is ensured by isolating the nucleic acids from thesample, however, isolation is optional (methods for amplifying nucleicacids, e.g., by PCR from whole cells are known and appropriate). Avariety of techniques for extracting nucleic acids from biologicalsamples are known in the art. For example, see those described inRotbart et al., 1989, in PCR Technology (Erlich ed., Stockton Press, NewYork) and Han et al., 1987, Biochemistry 26:1617-1625. The methodsdescribed by Fries et al., Am. J. Med. Genet., 46:363-368 (1993), arealso useful.

Nucleic acids are isolated from biological samples from patients, andfrom cell culture. The culture of cells used in conjunction with thepresent invention, including cell lines and cultured cells from tissueor blood samples, including stem cells is well known in the art.Freshney (Culture of Animal Cells, a Manual of Basic Technique, thirdedition Wiley-Liss, New York (1994)) and the references cited thereinprovides a general guide to the culture of cells. See also, Kuchler etal. (1977) Biochemical Methods in Cell Culture and Virology, Kuchler, R.J., Dowden, Hutchinson and Ross, Inc, and Inaba et al. (1992) J. Exp.Med. 176, 1693-1702.

When the sample contains a small number of cells, extraction may beaccomplished, e.g., by methods as described in Higuchi, “Simple andRapid Preparation of Samples for PCR”, in PCR Technology, Ehrlich, H. A.(ed.), Stockton Press, New York, which is incorporated herein byreference.

A relatively easy procedure for extracting DNA for amplification is a“salting out” procedure adapted from the method described by Miller etal., Nucleic Acids Res., 16:1215 (1988)

Kits are also commercially available for the extraction ofhigh-molecular weight (i.e., genomic) DNA. These kits include GenomicIsolation Kit A.S.A.P. (Boehringer Mannheim, Indianapolis, Ind.),Genomic DNA Isolation System (GIBCO BRL, Gaithersburg, Md.), Elu-QuikDNA Purification Kit (Schleicher & Schuell, Keene, N.H.), DNA ExtractionKit (Stratagene, La Jolla, Calif.), TurboGen Isolation Kit (Invitrogen,San Diego, Calif.), and the like. Use of these kits according to themanufacturer's instructions is generally acceptable for purification ofDNA when practicing the methods of the present invention.

The nucleic acids embracing the SNPs are typically amplified whendetermining whether a SNP is present in a sample. In a preferredembodiment, amplification is performed by the PCR method. The PCRprocess is well known in the art (see, U.S. Pat. Nos. 4,683,195;4,683,202; and 4,965,188. Strand separation may be induced by ahelicase, for example, or an enzyme capable of exhibiting helicaseactivity. For example, the enzyme RecA has helicase activity in thepresence of ATP. The reaction conditions suitable for strand separationby helicases are known in the art (see Kuhn Hoffman-Berling, 1978,CSH-Quantitative Biology 43:63-67; and Radding, 1982, Ann. Rev. Genetics16:405-436, both of which are incorporated herein by reference).

Template-dependent extension of primers in PCR is catalyzed by apolymerizing agent in the presence of adequate amounts of fourdeoxyribonucleoside triphosphates (typically dATP, dGTP, dCTP, and dTTP)in a reaction medium comprised of the appropriate salts, metal cations,and pH buffering system. Suitable polymerizing agents are enzymes knownto catalyze template-dependent DNA synthesis. In some instances,SNP-encoding RNA may be used as the initial template for primerextension is RNA. Polymerizing agents suitable for synthesizing acomplementary, DNA (cDNA) sequence from the RNA template are reversetranscriptase (RT), such as avian myeloblastosis virus RT, Moloneymurine leukemia virus RT, or Thermus thermophilus (Tth) DNA polymerase,a thermostable DNA polymerase with reverse transcriptase activitymarketed by Roche Molecular Systems. When RNA is amplified, an initialreverse transcription (RT) step is carried out to create a DNA copy(cDNA) of the RNA. PCT patent publication No. WO 91/09944, publishedJul. 11, 1991, incorporated herein by reference, describeshigh-temperature reverse transcription by a thermostable polymerase thatalso functions in PCR amplification. High-temperature RT providesgreater primer specificity and improved efficiency. A “homogeneousRT-PCR” in which the same primers and polymerase suffice for both thereverse transcription and the PCR amplification steps, and the reactionconditions are optimized so that both reactions occur without a changeof reagents is also available. Thermus thermophilus DNA polymerase, athermostable DNA polymerase that can function as a reversetranscriptase, is used for all primer extension steps, regardless oftemplate. Both processes can be done without having to open the tube tochange or add reagents; only the temperature profile is adjusted betweenthe first cycle (RNA template) and the rest of the amplification cycles(DNA template).

Those skilled in the art will know that the PCR process is most usuallycarried out as an automated process with a thermostable enzyme. In thisprocess, the temperature of the reaction mixture is cycled through adenaturing region, a primer annealing region, and an extension reactionregion. Alternatively, the annealing and extension temperature can bethe same. Reverse transcriptase-PCR uses such a two-step temperaturecycling. A machine specifically adapted for use with a thermostableenzyme is commercially available from Roche Molecular Systems.

Those practicing the present invention should note that, although thepreferred embodiment incorporates PCR amplification, amplification oftarget sequences it a sample may be accomplished by any known method,such as ligase chain reaction (LCR), transcription amplification, andself-sustained sequence replication, each of which provides sufficientamplification so that the target sequence can be detected by nucleicacid hybridization to a probe. Persons of skill will appreciate that inmethods such as LCR, primers that are complementary to the specificpolymorphism or mutation are used. In this instance amplification occurswhen the polymorphism (i.e., point mutation) is present in the nucleicacid sample.

Alternatively, methods that amplify the probe to detectable levels canbe used, such as replicase amplification. The term “probe” encompasses,inter alia, the sequence specific oligonucleotides used in the aboveprocedures; for instance, the two or more oligonucleotides used in LCRare “probes” for purposes of the present invention, even though someembodiments of LCR only require ligation of the probes to indicate thepresence of an allele.

Examples of techniques sufficient to direct persons of skill throughsuch in vitro amplification methods, including the polymerase chainreaction (PCR) the ligase chain reaction (LCR), Q.beta.-replicaseamplification and other RNA polymerase mediated techniques (e.g., NASBA)are found in Berger and Kimmel, Guide to Molecular Cloning Techniques,Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif.(Berger); Sambrook at al. (1989) Molecular Cloning—A Laboratory Manual(2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring HarborPress, NY, (Sambrook); and Current Protocols in Molecular Biology, F. M.Ausubel et al., ads., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1994Supplement) (Ausubel), and in Mullis et al., (1987) U.S. Pat. No.4,683,202; PCR Protocols A Guide to Methods and Applications (Innis etal. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim &Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991)3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173;Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell etal. (1989) J. Clin. Chem. 35, 1826; Landegren et al., (1988) Science241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu andWallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117, andSooknanan and Malek (1995) Biotechnology 13: 563-564. Improved methodsof cloning in vitro amplified nucleic acids are described in Wallace etal., U.S. Pat. No. 5,426,039.

The present invention provides, inter alia, a polymorphic forms ofCAIX9. The polymorphisms can be detected by a variety of amplificationtechniques, preferably PCR as described supra. To detect a polymorphismby PCR, the PCR reaction is performed in the presence of primers thatare complimentary to opposite strands of the genomic DNA, wherein thecomplementary sequences are located on either side of the pointmutation. The precise sequences recognized by the primers are notcritical. Typically, any pair of primers can be used as long as they (1)bracket the polymorphism, (2) are reasonably near to the polymorphism(while the primer binding sequence may be as far from the polymorphismas can support a PCR reaction, i.e., 1 to about 10 kb, it is preferablethat the binding sequence be within about 500 nucleotides or less, andmore preferable that the binding sequence be within 100 nucleotides ofthe SNP site to be assayed), and (3) bind the primers with an adequatedegree of specificity. It is preferable that the sequence be unique tothe gene of interest. Such sequences are identified by comparingsequences as described herein. Smaller primers have a higher probabilityof recognizing sites outside of the desired binding site, whereas verylarge primers are more expensive to make; generally, a primer of about15-20 nucleotides is adequate, and therefore preferred.

Exemplary primers used herein are found in Table 1.

The present invention also provides kits for the detection of geneticpolymorphisms or mutations associated with the disclosed SNPs. The kitscomprise a vial containing amplification primers that span a SNP. Thekits optionally contain a vial containing a thermostable polymerase,genetic size markers for gels, amplification reagents, instructions andthe like. The kit may also contain antibodies specific for CAIX protein.

A variety of methods can be employed to analyze the nucleotide sequenceof the amplification products. Several techniques for detecting pointmutations following amplification by PCR have been described in Chehabet al., Methods in Enzymology, 216:135-143 (1992); Maggio et al., Blood,81(1):239-242 (1993); Cai and Kan, Journal of Clinical Investigation,85(2):550-553 (1990) and Cai et al., Blood, 73:372-374 (1989).

One particularly useful technique is analysis of restriction enzymesites following amplification. In this method, amplified nucleic acidsegments are subjected to digestion by restriction enzymes.Identification of differences in restriction enzyme digestion betweencorresponding amplified segments in different individuals identifies apoint mutation. Differences in the restriction enzyme digestion iscommonly determined by measuring the size of restriction fragments byelectrophoresis and observing differences in the electrophoreticpatterns. Generally, the sizes of the restriction fragments isdetermined by standard gel electrophoresis techniques as described inSambrook, and, e.g., in Polymeropoulos et al., Genomics, 12:492-496(1992).

Another useful method of identifying point mutations in PCRamplification products employs oligonucleotide probes specific fordifferent sequences. The oligonucleotide probes are mixed withamplification products under hybridization conditions. Probes are eitherRNA or DNA oligonucleotides and optionally contain not only naturallyoccurring nucleotides but also analogs such as digoxygenin dCTP, biotindCTP, 7-azaguanosine, azidothymidine, inosine, or uridine. The advantageof using nucleic acids comprising analogs include selective stability,resistance to nuclease activity, ease of signal attachment, increasedprotection from extraneous contamination and an increased number ofprobe-specific colored labels. For instance, in preferred embodiments,oligonucleotide arrays are used for the detection of specific pointmutations as described below.

Probes are typically derived from cloned nucleic acids, or aresynthesized chemically. When cloned, the isolated nucleic acid fragmentsare typically inserted into a replication vector, such as lambda phage,pBR322, M13, pJB8, c2RB, pcos1EMBL, or vectors containing the SP6 or 17promoter and cloned as a library in a bacterial host. General probecloning procedures are described in Arrand J. E., Nucleic AcidHybridization A Practical Approach, Hames B. D., Higgins, S. J., Eds.,IRL Press 1985, pp. 17-45 and Sambrook, J., Fritsch, E. F., Maniatis,T., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press,1989, pp. 2.1-3.58, both of which are incorporated herein by reference.

Oligonucleotide probes and primers are synthesized chemically with orwithout fluorochromes, chemically active groups on nucleotides, orlabeling enzymes using commercially available methods and devices likethe Model 380B DNA synthesizer from Applied Biosystems, Foster City,Calif., using reagents supplied by the same company. Oligonucleotidesfor use as probes, e.g., in in vitro amplification methods, or for useas gene probes are typically synthesized chemically according to thesolid phase phosphoramidite triester method described by Beaucage andCaruthers (1981), Tetrahedron Letts., 22(20):1859-1862, e.g., using anautomated synthesizer, as described in Needham-VanDevanter et al. (1984)Nucleic Acids Res., 12:6159-6168. Oligonucleotides can also be custommade and ordered from a variety of commercial sources known to personsof skill. Purification of oligonucleotides, where necessary, istypically performed by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson and Regnier (1983) J.Chrom. 255:137-149. The sequence of the synthetic oligonucleotides canbe verified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enzymology 65:499-560.

Oligonucleotide probes and primers are selected using commerciallyavailable computer programs to compare known DNA sequences from genesequences found in gene libraries, such as Genebank and EMBL, and thesequences described herein. The programs identify unique nucleotidesequences within the gene of interest. One such program is Eugene.Oligonucleotide sequences for PCR of a unique genomic DNA such as achromosome subsequence are chosen optimally by choosing sequencesaccording to previously established protocols or by computer programsthat choose the degree of homology desired along with the length of theprobe. Sequences are chosen to avoid technical problems such as primerdimers resulting from amplification of hybridized primers.

Primers and probes are optionally labeled with fluorophores or enzymesthat generate colored products. This allows simultaneous use of probesto different DPDD-related polymorphisms or mutations. Identification ofhybridization of a specifically labelled primer provides a means fordetermining which polymorphism or mutation is present in the nucleicacid of the sample. The primers used in the assay are labeled with morethan one distinguishable fluorescent or pigment color. Primers arelabeled with Texas red, rhodamine and its derivatives, fluorescein andits derivatives, dansyl, umbelliferone and the like or with horse radishperoxidase, alkaline phosphatase, biotin, avidin, or the like.

Primers and probes are labeled directly or indirectly. The commonindirect labeling schemes covalently bind a ligand to the nucleotide andprepare labeled probe by incorporating the ligand using random primingor nick translation. The ligand then binds an ant-ligand which iscovalently bound to a label. Ligands and anti-ligands vary widely. Whena ligand has an anti-ligand, e.g., biotin, thyroxine, or cortisol, theligand is used in conjunction with the labelled naturally-occurringanti-ligand. Alternatively, a hapten or antigen may be used incombination with an antibody, which is optionally labeled.

Sequence specific oligonucleotide probes hybridize specifically with aparticular segment of the target polymorphism or mutation amplificationproducts and have destabilizing mismatches with the sequences from otherpolymorphisms or mutations. Under sufficiently stringent hybridizationconditions, the probes hybridize specifically only to exactlycomplementary sequences. The stringency of the hybridization conditionscan be relaxed to tolerate varying amounts of sequence mismatch.Detection of the amplified product utilizes this sequence-specifichybridization to insure detection of only the correct amplified target,thereby decreasing the chance of a false positive caused by the presenceof homologous sequences from related polymorphisms or mutations.

Specific CAIX polymorphisms or mutations are also identified bysequencing the amplification products or restriction fragments thereof.Sequencing is performed by a variety of methods well known in the art.For example, the sequence of the amplified nucleic acid segments may bedetermined by the Maxam-Gilbert chemical degradation method as describedin Sambrook. Generally, Sanger dideoxy-mediated sequencing is employedas described in Sambrook, or sequencing by hybridization is performed asdescribed below.

In one preferred class of embodiments, the SNP is detected byhybridization of amplification products which include the splicing siteto oligonucleotide arrays which discriminate single base-pairmismatches. In this embodiment, primers are used to amplify a SNP in aPCR reaction, resulting in PCR amplicons which comprise the SNP. Thesequence of the entire PCR amplicon, or any subsequence thereof can bedetermined by labeling the PCR amplicon (typically with biotin or afluorescent label) and hybridization to an array of oligonucleotideprobes. In these hybridization methods single base pair mismatches inlabeled nucleic acids to probes in the array are distinguished.

Preferably in this class of embodiments, the oligonucleotide arrays aredesigned to sequence nucleic acids at the SNP. More preferably, thearrays are designed to discriminate whether a particular nucleotide isaltered relative to the wild-type sequence. This is done by constructingan array with two or more oligonucleotide probe sets which differ by asingle nucleotide. Hybridization to the known probe sequence by a targetnucleic acid under conditions where a single mismatch does not bindindicates the presence of a fully complementary nucleic acid.

Sequencing by hybridization to arrays of oligonucleotides is describedin U.S. Pat. No. 5,202,231, to Drmanac et al. and, e.g., in Drmanac etal. (1989) Genomics 4:114-128. Methods of constructing and designingarrays for sequencing and detection of single nucleotide alterations isknown in the art. The development of very large scale immobilizedpolymer synthesis (VLSIPS™) technology provides methods for arranginglarge numbers of oligonucleotide probes for the detection and sequencingof nucleic acids in very small arrays. See, WO 90/15070 and 92/10092;Pirrung et al., U.S. Pat. No. 5,143,854 (see also PCT Application No. WO90/15070); McGall et al., U.S. Pat. No. 5,412,087; and U.S. Pat. No.5,384,261. See also, Fodor et al. (1991) Science, 251: 767-777 andSheldon et al. (1993) Clinical Chemistry 39(4): 718-719. Theoligonucleotide arrays are typically placed on a solid surface such as aglass slide with an area less than 1 inch squared, although much largersurfaces are optionally used.

Mechanical and light directed oligonucleotide array construction methodsare used for the construction of oligonucleotide arrays. Light directedmethods are the most common, and are found, e.g., in U.S. Pat. No.5,143,854. The light directed methods discussed in the '854 patenttypically proceed by activating predefined regions of a substrate orsolid support and then contacting the substrate with a preselectedmonomer solution. The predefined regions are activated with a lightsource, typically shown through a photolithographic mask. Other regionsof the substrate remain inactive because they are blocked by the maskfrom illumination. Thus, a light pattern defines which regions of thesubstrate react with a given nucleic acid reagent. By repeatedlyactivating different sets of predefined regions and contacting differentreagent solutions with the substrate, a diverse array ofoligonucleotides is produced on the substrate. Other steps, such aswashing unreacted reagent solutions from the substrate, are used asnecessary.

The surface of a solid support is typically modified with linking groupshaving photolabile protecting groups and illuminated through aphotolithographic mask, yielding reactive hydroxyl groups in theilluminated regions. For instance, during oligonucleotide synthesis, a3′-O-phosphoramidite (or other nucleic acid synthesis reagent) activateddeoxynucleoside (protected at the 5′-hydroxyl with a photolabile group)is then presented to the surface and coupling occurs at sites that wereexposed to light in the previous step. Following capping, and oxidation,the substrate is rinsed and the surface illuminated through a secondmask, to expose additional hydroxyl groups for coupling. A second5′-protected, 3′O-phosphoramidite activated deoxynucleoside (or othermonomer as appropriate) is then presented to the resulting array. Theselective photodeprotection and coupling cycles are repeated until thedesired set of oligonucleotides (or other polymers) is produced.

The PCR amplicons detected on the arrays are labeled with a compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,or chemical means. For example, useful labels include ³²P, ³⁵S,fluorescent dyes, chromophores, electron-dense reagents, enzymes (e.g.,as commonly used in an ELISA), biotin, dioxigenin, or haptens andproteins for which antisera or monoclonal antibodies are available. Inpreferred embodiments, the label is detectable spectroscopically, i.e.,is chromogenic. Suitable chromogens include molecules and compoundswhich absorb light in a distinctive range of wavelengths so that a colormay be observed, or emit light when irradiated with radiation of aparticular wavelength or wavelength range (e.g., a fluorescent label).

EXAMPLES

Methods of immunotherapy using IL-2 and/or interferon alpha are alsoknown in the art. (see, Fyfe et al., Journal of Clinical Oncology, Vol13, 688-696 (1995) and Rosenberg et al., Annalso of Surgery, Vol. 228,No. 3, 307-319 (1998) which are incorporated herein by reference.

In all the above aspects and embodiments of the invention having SNPsubject matter, in some embodiments, the SNP is preferably SNP1(rs12553173 (c.249T>C)).

The following examples are offered by way of illustration and notlimitation. One of skill will readily recognize a variety of parametersand conditions which can be changed or modified to yield essentiallyidentical results.

The Applicants assessed the frequency of Carbonic Anhydrase 9 (CA9)single nucleotide polymorphisms (SNPs) and mutations and theirassociation with CAIX protein expression, response to IL-2 and overallsurvival in 54 Caucasian patients with metastatic clear cell renal cellcarcinoma (MRCC). Genomic DNA was extracted from frozen tumor samples.Seven amplimers covering the whole coding sequence of the CA9 gene weresynthesized by PCR and sequenced. The monoclonal antibody M75 was usedto evaluate CAIX protein expression immunohistochemically. Associationsof SNPs with clinicopathological variables and CAIX expression wereassessed with Fisher's Exact tests and Kruskal-Wallis tests,respectively. CA9 reference SNP (rs) 2071676 was found in 59%,rs12553173 in 15%, rs3829078 in 11% and rs1048638 in 33% of thepatients. The deletion c.376del393 was observed in two patients. CAIXexpression was high (>85%) in 65% and low in 35% of patients. None ofthe SNPs was associated with CAIX expression, but a trend was observedfor the presence of a SNP at rs12553173 (high CAIX: 88% vs. 61%,p=0.145). Patients with the C allele variant of rs12553173 had improvedoverall survival compared to those without (median survival: 27.3 vs.13.6 months, p=0.0431) and a greater likelihood of response to IL-2 (57%vs. 22%, p=0.081) No other SNPs was associated with overall survival orresponse to IL-2. Likewise, high CAIX expression was associated withlonger median survival (25.5 vs. 8.5 months, p<0.0001) and a greaterIL-2 response rate (37% vs. 8%, p=0.070). In a multivariate Coxproportional hazards model, both C allele variant of CA9 SNP rs12553173and CAIX expression were retained as independent prognostic factors ofoverall survival. The details of this work are next provided.

Patients and Methods

The study included 54 consecutive Caucasian patients, who underwentradical nephrectomy with regional lymph node dissection for sporadic,unilateral, clear cell MRCC. Only Caucasian patients were chosen for thestudy, since the frequencies of CA9 SNPs differ among the races and racehas been shown to be a prognostic indicator for MRCC [24]. Moreover,only patients with clear cell MRCC were included, since CAIX is mostsignificantly associated with the pathobiology and prognosis in thissubtype [25, 26].

Age, gender, ECOG performance status [27], 2002 T, N, and M stage [28],and overall survival were collected for each case. Hematoxylin & Eosin(H&E) slides were reviewed by one anatomical pathologist (DS) to confirmthe histological subtype and to re-grade tumors according to Fuhrmancriteria [29]. The study protocol was approved by the institutionalreview board.

Genomic DNA was extracted from 50 mg frozen tissue sections using QIAampDNA minikit (Qiagen Inc., Valencia, Calif.). DNA quantity and qualitywere estimated by optical density (OD 260/280) measurement and 0.8%agarose gel electrophoresis using standard protocols.

All eleven CA9 exons and flanking intronic regions were PCR amplified byspecific primer pairs (Table 1). We amplified 50 to 150 ng of tumor DNAin 50 μA, with a final MgCl₂ specific for each amplification, 100 ng oftemplate DNA, 1× reaction buffer, 0.2 mM of each nucleotide, 30 μmol ofprimers and 0.3 U of DNA polymerase (Platinum Taq, Invitrogen, Carlsbad,Calif.). PCR reactions were carried out for 35 cycles with denaturationat 94° C. for 1 min, annealing at 60° C. for 1 min and extension at 72°C. for 1 min. Forward and reverse automatic sequencing was performedusing BigDye Terminator v1.1 Cycling Sequencing kit on a 3730 DNAAnalyzer (Applied Biosystems, Foster City, Calif.). All SNPs andmutations were confirmed in a second round of PCR and sequencingreactions.

CAIX Protein Expression

CAIX protein expression of the primary tumor was evaluated byimmunohistochemistry using the tissue microarray technique, as describedpreviously [25]. In brief, three core tissue biopsies of the tumor andone core tissue biopsy from normal renal tissue were taken from eachparaffin-embedded specimens and precisely arrayed using a custom-builtinstrument [30]. A Dako Envision staining system (Dako, Carpinteria,Calif.) and the mouse monoclonal antibody MN75 at a 1:10,000 dilution (agift from Dr. Eric Stanbridge, University of California-Irvine) wereused for the staining Semi-quantitative assessment of CAIX staining wasperformed by one anatomical pathologist (DS) blinded to pathologicalvariables, CA9 status and survival. Expression was evaluated as thepercentage of the entire tumor sample that stained positive for CAIX.

Analyses were all performed using R v2.4. Associations of SNPs withclinicopathological variables and CAIX expression were assessed withFisher's Exact tests and Kruskal-Wallis tests, respectively.Kaplan-Meier curves were generated to estimate the overall survivorfunctions, which were compared using log-rank tests. A multivariate Coxproportional hazards regression model was fit to identify factorsindependently associated with overall survival.

Results

Patient Population

There were 42 men (78%) and 12 women (22%), who were diagnosed withclear cell MRCC with a median age of 64 years (range 34-78). An ECOG PSof >1 was assigned to 41 patients (76%). Pathological examination showeda T1, T2, T3, and T4 tumor in 7 (13%), (9%), 36 (67%), and 6 (11%)patients, respectively. Involvement of the regional nodes (N+) wasobserved in 14 patients (26%). Fifty percent of the tumors were highgrade (Grade 3 or 4).

CA9 SNPs

Sequencing of CA9 in our patients showed SNPs in exon 1, exon 7 and the3′ UTR region, while all other nine exons had wild type sequences (Table2, Table 3). Most SNPs were noted in exon 1. On position 201 of thecDNA, guanine was replaced with adenine (c.201G>A), referring toreference SNP (rs)2071676 of the SNP NCBI database(http://www.ncbi.nlm.nih.gov/SNP/snp reficgi?rs=2071676). A SNP atrs2071676 was seen in 32 tumors (59%), of which 21 were heterozygous and11 homozygous. The second most frequent variant was the synonymous SNPrs12553173 (c.249T>C), which was detected in 8 tumors (15%; 7heterozygous, 1 homozygous). In exon 7, we noted the SNP rs3829078(c.1081A>G) in 6 cases (5 heterozygous, 1 homozygous). The SNP rs1048638(c.1584C>A), located in the non-coding 3′ UTR flanking region, wasobserved in 18 cases (33%), of which 16 were heterozygous and 2homozygous. Interestingly, all cases with rs1048638 had the wild typesequence for rs3829078. All 11 patients with homozygous rs2071676 hadthe wild type sequence for rs12553173. CA9 SNPs, frequency, positions,function, and subsequent amino acid changes are summarized in Table 3.In addition to SNPs, we observed the novel deletion c.376del393 in twopatients.

CAIX Protein Expression

CAIX staining was seen in 52 of the 54 tumors (96%), and was notobserved in normal renal tissue as previously described. Using the 85%expression cut-point defined by Bui et al. [25], expression wasconsidered high (>85%) in 35 tumors (65%) and low (<85%) in 19 tumors(35%).

None of the SNPs were significantly associated with CAIX expression.However, a trend was observed for the SNP rs12553173 (c.249T>C): 88% ofthe tumors with rs12553173 showed high CAIX expression in contrast toonly 61% of tumors with the wild type sequence at this location.However, due to small sample size, this difference did not quite reachstatistical significance (p=0.145). Likewise, CAIX expression was higherwhen expression levels were assessed as a continuous variable (meanexpression±SE: 88.2±11.7% vs. 78.1±4.9%, p=0.100).

Association with Overall Survival and Response to IL-2

At the time of analysis, 49 of 54 patients (91%) had died. The mediansurvival time was 15.7 months. Patients with the C allele variant at SNPrs12553173 (c.249T>C) had improved overall survival compared to thosewithout (median survival: 27.3 vs. 13.6 months, p=0.0431). All othervariants were not associated with overall survival. Likewise, high CAIXexpression was associated with longer median survival (25.5 vs. 8.5months, p<0.0001), as published previously [25, 31, 32]. We fit amultivariate Cox proportional hazards model to identify factors thatwere independently associated with overall survival. In this model, ECOGPS, T stage, CAIX expression and rs12553173 were identified asindependent prognostic factors (Table 4).

Of the 54 patients, 43 (79%) received IL-2 based immunotherapy postnephrectomy. For the assessment of response, complete responses (CR) andpartial responses (PR) were pooled into one group and compared tonon-responders (stable disease, progressive disease). Four out of 7(57%) patients with rs12553173 who had received IL-2 responded comparedwith 8 of 36 (22%) without rs12553173 (p=0.081). In contrast, rs2071676(p=0.168), rs3829078 (p=0.123), rs1048638 (p=0.484) were all notassociated with response to IL-2. In terms of CAIX protein expression,the response rate to IL-2 was 37% ( 11/30) in patients with high and 8%( 1/13) in patients with low CAIX expression (p=0.070). All four CRswere observed in patients with high CAIX.

Discussion

We analyzed the CA9 gene coding sequence, CAIX protein expression andtheir association with survival and response to IL-2 in 54 Caucasianclear cell MRCC patients. We found 4 different SNPs in 2 of the 11 CA9exons and the 3′ UTR region, of which occurrence of rs12553173(c.249T>C, exon 1) was associated with improved overall survival andretained as an independent prognostic factor in multivariate analysis.Furthermore, presence of the variant rs12553173 yielded a greaterlikelihood of response to IL-2 based immunotherapy. As describedpreviously by us and others [25, 31, 32], high CAIX protein expressionwas associated with longer survival and a greater IL-2 response rate.

SNPs have been associated with survival in several cancer entities[1]-17], but to date not in RCC. Furthermore, the current study is thefirst showing that a synonymous SNP is associated with survival in anytype of cancer. As a synonymous CA9 SNP, rs12553173 does not alter thesequence of the CAIX protein. Until recently, synonymous SNPs wereregarded as non-relevant, because it was thought that they are not ableto affect expression and function of the related proteins. During thepast years, however, several groups have pointed out that occurrence ofsynonymous SNPs do have an impact on the occurrence and course ofdiseases [10]. Kimchi-Sarfaty et al. [33] studied the synonymous SNP1236C>T, the synonymous SNP 3435C>T and the non-synonymous 2677G>T SNPin the MDR1 gene, which plays a role in the development of resistance tochemotherapy. They found that the synonymous SNP 3435C>T is the keypolymorphism of this haplotype, and that the non-synonymous SNP alonehas no effect on the protein. A study of Laws et al. [34] showed that asynonymous SNP in the apoE gene carries a higher risk to developAlzheimer. Capon et al. [35] found a synonymous SNP in thecorneodesmosin gene, which may lead to psoriasis. The mechanisms howsynonymous SNPs affect the protein are not completely understood.Current findings suggest that a synonymous SNP can change the amount,the structure and/or the function of a protein by three keymechanisms: 1) impacting mRNA structure and stability, 2) impactingkinetics of translation, and 3) alternating splicing [33]. Whichmechanisms are responsible for improved survival of patients withrs12553173, remains elusive.

The CA9 gene encodes for the CAIX protein, which is widely regarded asone of the most significant molecular markers in MRCC [36]. Severalstudies have investigated the role of CAIX protein expression in MRCCand have consistently shown that high tumoral CAIX expression isassociated with better prognosis and a greater likelihood of response toIL-2 based immunotherapy [37, 38]. This study represents yet anotherconfirmation of the importance of CAIX protein in predicting survivaland IL-2 response in MRCC. We further hypothesized that CA9 SNPs wouldbe associated with CAIX protein expression. However, this associationwas not clearly observed. More importantly, both CA9 SNP rs12553173 andCAIX expression were complementary in predicting prognosis and bothretained as independent prognostic factors of overall survival.

However, they did not correlate their findings with survival. Thecurrent study serves as another example that genetic (rs12553173) andprotein information (CAIX expression) can predict prognosis of MRCC andshould be integrated into future prognostic models and clinical carealgorithms.

This study has several limitations. We investigated CA9 SNPs in tumortissue and not in the blood, which was not possible because of theretrospective nature of this study. However, the SNPs that were detectedare well described, and it is very unlikely that sequencing of CA9 inleukocytes would have yielded other results.

TABLE 1 Primers used in the study SEQ Annealing ID Temperature Exon(s)Direction Primer sequence NO: (° C.) 1 Forward 5′- -3′gactttggctccatctctgc 4 60 Reverse 5′- -3′ ctggaacctggatttggaga 5 2-3Forward 5′- -3′ cgtttgtgacatcgttttgg 6 60 Reverse 5′- -3′gccccatccccaagtctc 7 4-5 Forward 5′- -3′ ctcacttgcctctccctacg 8 60Reverse 5′- -3′ atagagtccgggaggagcat 9 6 Forward 5′- -3′agctgaggaatgggagaggt 10 60 Reverse 5′- -3′ cagacctgaagctccaaagg 11 7Forward 5′- -3′ aagctttaagggggtgcaat 12 60 Reverse 5′- -3′ccactgtgtccacacacacc 13 8-9 Forward 5′- -3′ cacccacactgtccactgac 14 60Reverse 5′- -3′ aaaaggagagggagcagagg 15 10-11 Forward 5′- -3′ggcaggtgttgaggaactct 16 60 Reverse 5′- -3′ ggggaacaaaggtgactaaca 17

TABLE 2 Patient and tumor characteristics, CAIX expression and CA9 genestatus in 54 patients with metastatic clear cell RCC No. of CAIX CA9 CA9CA9 CA9 Gender, ECOG TNM stage, M1 FU months, expression Exon 1 Exon 1Exon 7 Exon 11 Age PS Fuhrman grade sites status % c.201G > A c.249T > Cc.1081A > G c.1584C > A M, 69 2 T3 N0 M1 G3 2 2, dead 46.5 Hetero heteroM, 51 1 T3 N2 M1 G3 1 95.3, alive 86.7 F, 64 2 T3 N0 M1 G2 3 19.9, dead70.0 M, 71 1 T3 N0 M1 G3 2 15.1, dead 100.0 Homo M, 78 1 T3 N0 M1 G2 227.3, dead 100.0 Hetero hetero hetero M, 57 1 T3 N0 M1 G2 2 35.5, alive100.0 homo hetero M, 75 1 T3 N0 M1 G2 1 10.7, dead 100.0 M, 69 1 T2 N0M1 G3 1 61.5, dead 100.0 F, 46 1 T1 N0 M1 G2 2 23.3, dead 80.0 Homohetero M, 65 1 T1 N0 M1 G3 1 34.8, dead 100.0 Hetero M, 55 0 T3 N0 M1 G23 75.5, alive 100.0 M, 68 1 T1 N0 M1 G2 1 122.9, dead 100.0 hetero M, 672 T4 N2 M1 G3 2 1.8, dead 90.0 Homo hetero F, 67 1 T1 N0 M1 G2 1 20.8,dead 0.0 Homo M, 58 1 T3 N0 M1 G3 2 10.4, dead 100.0 M, 65 1 T3 N0 M1 G21 8, dead 100.0 F, 52 0 T3 N0 M1 G2 1 15.8, dead 100.0 Homo M, 71 1 T4N1 M1 G4 3 10.2, dead 85.0 Hetero hetero M, 66 0 T4 N0 M1 G3 2 62.3,dead 100.0 Hetero hetero M, 52 1 T3 N0 M1 G2 2 21.8, dead 100.0 Heterohetero M, 57 0 T3 N0 M1 G2 2 39.7, dead 26.7 Hetero hetero M, 63 0 T3 N0M1 G2 2 81.6, alive 100.0 Homo hetero M, 65 0 T3 N0 M1 G2 1 114.1, dead100.0 Hetero hetero hetero M, 66 1 T3 N0 M1 G3 1 75.5, dead 100.0 heterohetero F, 68 1 T3 N2 M1 G2 1 2.9, dead 70.0 Hetero hetero M, 70 1 T1 N0M1 G3 2 108.1, dead 98.0 F, 74 1 T3 N0 M1 G3 1 24.4, dead 100.0 Heterohetero hetero M, 66 1 T3 N2 M1 G3 2 4.3, dead 100.0 hetero M, 48 1 T3 N0N1 G3 2 2.3, dead 1.7 Homo hetero M, 68 0 T3 N0 M1 G3 1 40.6, dead 100.0Homo M, 59 1 T4 N0 M1 G2 1 1.4, dead 100.0 F, 66 1 T3 N0 M1 G2 4 5.8,dead 96.7 Hetero hetero M, 48 1 T4 N0 M1 G3 2 2.8, dead 83.3 M, 58 1 T3N0 M1 G3 2 8.5, dead 41.3 Hetero M, 40 1 T2 N0 M1 G2 1 12.2, dead 70.0Homo hetero M, 34 1 T3 N2 M1 G3 2 13.2, dead 6.1 Hetero hetero homo F,53 1 T1 N1 M1 G4 3 10.8, dead 0.0 Hetero hetero M, 74 0 T3 N0 M1 G2 224.9, dead 92.5 Hetero hetero M, 71 0 T3 N0 M1 G2 1 31.6, dead 100.0Homo homo M, 74 2 T1 N0 M1 G2 1 13.6, dead 11.0 homo F, 69 1 T2 N0 M1 G11 23.6, dead 100.0 M, 50 1 T4 N1 M1 G2 2 2.7, dead 0.8 F, 48 1 T2 N2 M1G3 1 85.6, dead 100.0 F, 64 1 T3 N2 M1 G3 2 2, dead 100.0 Hetero M, 68 1T3 N1 M1 G3 2 0.9, dead 77.6 Hetero M, 62 0 T3 N0 M1 G3 2 10.9, dead100.0 Hetero M, 54 1 T3 N0 M1 G2 2 25.5, dead 100.0 M, 47 1 T3 N0 M1 G41 12, dead 100.0 Hetero F, 63 1 T3 N0 M1 G2 1 4.7, dead 65.0 M, 68 0 T2N0 M1 G3 2 5.6, dead 79.0 M, 51 0 T3 N2 M1 G3 2 90.8, alive 100.0 Homohetero M, 59 1 T3 N1 M1 G3 2 26.9, dead 100.0 Hetero M, 51 1 T3 N2 M1 G23 2.4, dead 25.0 M, 54 0 T3 N0 M1 G2 1 23.1, dead 100.0 Hetero hetero

TABLE 3 Multivariate Cox proportional hazards model. ECOG PS, T stage,CAIX expression and CA9 SNP c.249T > C were all retained as independentprognostic factors of overall survival. Categories HR 95.0% CI p-valueECOG PS ≧1 vs. 0 4.982 2.136 11.620 0.0002 T stage T¾ vs. T½ 4.338 1.84910.177 0.0007 N stage N+ vs. N0 0.615 0.275 1.378 0.2378 Fuhrman gradeG¾ vs. G½ 1.153 0.583 2.280 0.6820 Metastatic sites ≧2 vs. 1 1.040 0.5481.974 0.9036 CAIX expression Continuous 0.983 0.973 0.993 0.0005 CA9 SNPrs12553173 Yes vs. No 0.234 0.087 0.628 0.0039

In an independent group of additional ˜20 patients, preliminary studiescontinue to find that in patients with metastatic clear cell tumors,SNP1 is a significant predictor of survival in a multivariate model thatincludes the CAIX status and whether or not the patients receivedcytokine immunotherapy:

CC, M1 (n = 24) Variable HR HR low HR high p SNP1 0.163 0.035 0.7530.0201 IMMUNO 0.033 0.005 0.220 0.0004 CAIX 0.984 0.971 0.997 0.0135

TABLE 4 Variants, position, function, amino acid changes, and frequencyof the observed CA9 sequence variations. Data according to NCBI database(http://www.ncbi.nlm.nih.gov/projects/SNP/). cDNA position Contig andnucleotide mRNA dbSNP rs# dbSNP Protein Codon Current study, FrequencyExon position change position cluster id Function allele residueposition Hetero Homo Total 1 35664053 c.201G > A 139 rs2071676 Non-GTG > ATG Val[V] > Met[M] 1 0.39 0.20 0.59 synonymous 1 35664101c.249T > C 187 rs12553173 Synonymous TTG > CTG Leu[L] > Leu[L] 1 0.130.02 0.15 7 35669251 c.1081A > G 1019 rs3829078 Non- CAA > CGA Gln[Q] >Arg[R] 2 0.09 0.02 0.11 synonymous 11 35671122 C.1584C > A 1522rs1048638 3'UTR A/C — — 0.30 0.04 0.33

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What is claimed is:
 1. A method of treating a human patient havingmetastatic renal cell carcinoma (MRCC), said method comprising: (a)obtaining a biological sample containing Carbonic Anhydrase 9 (CA9)nucleic acid from the patient; (b) analyzing the biological sample todetermine whether the CA9 nucleic acid has a C allele at polymorphicsite rs12553173, thereby determining whether the patient has a C alleleat polymorphic site rs12553173; (c) determining that the patient has anincreased likelihood of responding to an IL-2 immunotherapy when thepatient has a C allele at polymorphic site rs12553173; and (d)administering the IL-2 immunotherapy to the patient having the C alleleat polymorphic site rs12553173, thereby treating the patient.
 2. Themethod of claim 1, wherein the MRCC is metastatic clear cell renal cellcarcinoma.
 3. The method of claim 1, wherein the nucleic acid is cDNA orRNA.
 4. The method of claim 1, wherein the nucleic acid is genomic DNA.5. The method of claim 1, wherein the biological sample is from a tumor.6. The method of claim 1, wherein the biological sample is from a renaltumor or a metastatic lesion derived from a renal tumor.
 7. The methodof claim 1, wherein the analyzing step comprises amplifying the CA9nucleic acid by polymerase chain reaction (PCR).